Mechanism Of Autophagy Mediating Microglial Polarization In An In Vitro Model Of Subarachnoid Hemorrhage

Objective:Previous studies have found that autophagy can affect the polarized phenotype of microglia in a variety of neurological disorders,but the relationship between autophagy and microglial polarization in the Subarachnoid hemorrhage(SAH)model is unclear.In this study,we intend to use hemoglobin to stimulate BV-2 cells to mimic SAH in vitro models to further explore the effect of autophagy on the polarization trend of microglia in the SAH microenvironment.Methods:Firstly,BV-2 cells were stimulated to simulate the SAH in vitro model using 25 u M hemoglobin(Hb),divided into control group and Hb(6,12 h,24 h and 48 h)groups,and LC3 and P62 protein contents were detected by western blotting to evaluate the autophagy level of microglia at different time points after modeling.Then,the above method was used again to make the mold,but the autophagy promoter rapamycin and the autophagy inhibitor 3-methyladenine(3-MA)or baflomycin(Baf A1)were used in the mold making process,and the in vitro model was divided into control group,Hb group,Hb + rapamycin group,Hb + 3-MA and Hb + Baf A1 group according to different intervention methods,and Western blotting and quantitative PCR were used to detect iNOS(M1 microglial marker),Arg-1(M2 microglial markers),and immunofluorescence was further used to detect CD86(M1 microglial marker)and CD206(M2 microglial marker).Results:1.Western blotting analysis showed that compared with the control group,the LC3II/I ratio of the Hb group was reduced,and the level of P62 protein was increased,which was the most obvious and statistically significant 24 h after molding.2.Western blotting and quantitative PCR analysis showed that compared with the control group,the level of Arg-1 in the Hb group was reduced and the level of iNOS was increased;Compared with the Hb group,the level of Arg-1 and iNOS in the Hb + 3-MA group(P<0.05)and Hb + Baf A1 group(P<0.05)were reduced,while the level of Arg-1and iNOS was increased in the Hb + rapamycin group(P<0.05).Compared with the Hb group,the ratio of CD206+Iba-1+/Iba-1+ in the Hb + 3-MA group(P<0.05)and the Hb+Baf A1 group(P<0.05)decreased,the ratio of CD86+Iba-1+/Iba-1+ increased,while the ratio of CD206+Iba-1+/Iba-1+ in the Hb + rapamycin group(P<0.05)increased and the ratio of CD86+Iba-1+/Iba-1+ decreased.Conclusion:In the in vitro subarachnoid hemorrhage model,Hb stimulation can lead to dynamic impairment of microglial autophagy flow;Promoting autophagy can polarize microglia to M2 type,while inhibition of autophagy can polarize microglia to M1 type.